The function of the Glgi complex will be studied in two polarized epithelial cell lines. The cell lines (MDCK and WIF-B) represent models of two epithelia (kidney tubule and hepatocyte) that use a different mechanism and/or site for protein and lipid sorting for delivery to specialized plasma membrane domains. The golgi complex plays an important role in this process. Malfunction of the sorting machinery in polarized epithelial cells has the potential to cause disease, since the integrity of these cells is required to define and maintain physiological compartments in the organism. Understanding the role of the Golgi complex in directing proteins and lipids their site of function is instrumental to understanding the disease process. The Golgi complex consists of at least three subcompartments: the stacks, and two pleomorphic compartments on either side, the cis Golgi network (CGN) and the trans Golgi network (TGN). The contribution of the lipid bilayer to Golgi structure and function in MDCK and WIF-B cells will be investigate. Distribution, structure and biosynthesis of the unusual phospholipid semilysobisphosphatidc acid (enriched in the CGN and TGN) will be determined, and its function will be addressed using perturbations. The glycosphingolipid and cholesterol content of Golgi subcompartments in control cells and in cells after various perturbations will be determined, since these lipids have been postulated to have a role in sorting in epithelial cells. In addition, since hepatocytes are believed to lack a direct pathway for delivery of newly synthesized proteins from the Golgi to the apical plasma membrane, the pathway by which newly synthesized sphingolipids are delivered to the apical plasma WIF-B cells will be investigated. The phosphatidylinositols are important for Golgi function in a role that is apparently distinct fromclassic signal transduction pathways. A defective phsphatidylinositol 4,5-bisphosphate 5-phosphatase localized to Golgi membranes was recently implicated in the oculocerebrorenal syndrome of Lowe, in which a subset of polarized cells is disfunctional. Golgi lipid composition (including phosphatidylinositol 4,5- bisphosphate) and potential protein sorting defects in MDCK cells lacking the 5-phosphatase will be assessed.